The pathologic proteolytic activation of digestive zymogens (primarily proteases such as chymotrypsinogen) within pancreatic acinar cells is a key step in initiating acute pancreatitis. Our laboratory examines extracellular factors and intracellular mechanisms responsible for this activation. Supraphysiologic (>10 fold than required for maximal secretion), but not physiologic concentrations of cholecystokinin (CCK), given in vivo cause zymogen activation and pancreatitis. Similarly treated isolated pancreatic acinar cells respond with zymogen activation and injury. Factors that can sensitize the acinar cell might convert the physiologic actions of CCK and other ligands to pathologic responses and may be relevant to the pathogenesis of acute pancreatitis. For example, experimental studies of pancreatitis induced by ischemia, bile salts, the CDE diet, pancreatic duct obstruction and ethanol suggest that such sensitization contributes to disease. During our past funding period we reported the sensitizing effects of cAMP agonists and alcohols on CCK-induced zymogen activation. We propose to continue examining the mechanisms of sensitization. Further, we have also found that the assembly and activation of a vacuolar ATPase (vATPase) is central to acinar cell zymogen activation. Indeed, it may be the final mediator of the zymogen activation response. It is relevant vATPase's containing distinct isoforms can be localized to specific organelles and activated by different mechanisms. Preliminary studies suggest that zymogen activation takes place in a compartment associated with a specific vATPase isoform (V0a2). Given the importance of the vATPase in this phenomenon and its role in other cell systems, it will be a major focus of our studies. The following specific aims will be pursued. 1) Define the vacuolar ATPase subunit composition, subcellular distribution, localization and assembly responses after cholecystokinin treatment in acini and in vivo using physiologic conditions and those that generate acute pancreatitis, 2) Examine the sensitizing effects alcohols and glucose on pathologic zymogen activation in the pancreatic acinar cell and vATPase activation and assembly, 3) Examine the effects of specific signaling pathways (cAMP, PKC, AMPK) on zymogen activation and vATPase assembly in intact cells and in a cell-free system and the effects of the vATPase on calcium signaling, 4) Develop new approaches to reduce vATPase activity using molecular and biochemical approaches.